The invention mainly, but not exclusively, relates to a simple, rapid and precise procedure for controlling the absolute number of cells (or other particles) in suspension found per unit volume of a sample.
The procedure of the invention makes use of a flow cytometer equipped with one or more lasers and is based on the use of a mixture of different populations of reference particles, in known quantities and proportions. The procedure is of use for research, diagnostic, prognostic purposes and to evaluate therapeutic protocols.
Enumeration of absolute counts of cells (or other particles) of a sample, represents information of utmost importance both in biomedical research and in clinical diagnostic laboratories.
Currently, a number of different methods are available for the enumeration of absolute cell counts present in a sample, the most accurate and precise of which use samples where the particles to be counted are in suspension. Of these, the most notable techniques employ two types of instruments: hematological analyzers and flow cytometers.
While the hematological analyzers employ volumetric methods for the counting of particles detected through the measurement of the impedance and more recently through the light scatter of a laser, the principles behind the enumeration of absolute counts of cells or other particles in flow cytometers are diverse and include various volumetric methods or the use of reference particles.
Though the flow cytometry volumetric methods for the enumeration of absolute counts of cells or other particles can only be applied in a limited number of instruments, with only two commercially available at present, methods based on the the use of reference particles can be applied to any flow cytometer independently of the manufacturer, including the most popular models.
Through this latter method, we can determine the number of cells (or other particles) present in a defined volume of a sample, adding a defined number of reference particles to a certain volume, also pre-determined, of the sample. The accuracy of the method depends on the preparation of the mixture of reference particles and the measurement of the volume of the sample, independently of the possible addition to the former mixture of variable volumes of other reagents that make measurements more difficult, such as monoclonal antibodies to identify the cells of interest or lysing solutions which specifically destroy non-nucleated cells (for example, the red blood cells in samples used to study leukocytes).
Without doubt, the most critical steps in this technique are
                1) the precise measurement of a determined volume of the sample.        2) the mixture with the sample of a precise number of reference particles.Currently, there are two ways to approach the latter question: i) add a known volume of the sample to tubes containing lyophilized reference microbeads—TRUCOUNT™ tubes—or, alternatively ii) to add to a tube containing a known volume of the sample, an accurately measured volume of a solution which contains, in suspension, a known number of particles or microbeads—FLOWCOUNT™ spheres. In the latter, it is assumed that the stock solution of FLOWCOUNT™ microbeads from which a precise volume is pipetted and which has been previously vortexed, contains a homogeneously distributed suspension of reference microbeads. At the same time, in both methods—FLOWCOUNT™ and TRUCOUNT™—it is assumed that during measurement no preferential selection exists either of the reference microbeads or of the cells (or other particles) present in the sample from which the count is to be made.        
In recent years, both methods for calculating the absolute count of particles have been adopted for use, initially by research laboratories but, subsequently, by clinical diagnostic laboratories too. Among other uses, they have been employed for the count of CD4+ T-lymphocytes in peripheral blood of individuals infected with human immunodeficiency virus (HIV), as well as of CD34+ stem and hematopoietic precursor cells in leukapheresis products in order to predict the success or failure of a prospective transplant. Preliminary studies have shown that disturbing levels of variability exist in these types of measurements which are usually reduced with the training of the personnel responsible for carrying out the technique.
Despite partial standardization of the techniques and methods described above, until now, in these types of measurements there has been no procedure described which would address the control of two significant variables:                1) the homogenous distribution of the reference particles, both in the stock solution and once mixed with the sample to be measured.        2) the selective acquisition of cells or reference particles during measurement in the flow cytometer.        